Apparatus for counting discrete microscopic particles



Feb. 5, 1963 NES ETA A. R. JO L 3,076,600 APPARATUS FOR COUNTING DISCRETE MICROSCOPIC PARTICLES Filed Sept. 3, 1954 2 Sheets-Sheet. 1

. I I l I I I I l I l I l l l l I I I I l I I I l I I A. R- JONES ETAL APPARATUS FOR COUNTING DISCRE MICROSCOPIC PARTICLES Feb. 5, 1963 Filed Sept. 3, 1954 lnveiii'ofls:

a I y fliau We? Jones n u n r mm II.\.IIIIIIIIII.I I IW q u m. QM .9 m lwi mm I n rIlII 5 a a mm .3 F q P L United States Patent 7 3,076,600 APPARATUS FOR COUNTING DISCRETE MICROSCOPIC PARTICLES Alan Richardson Jones, Wellesley Hills, and Frederick Brech, Needham, Mass., assignors to Jarrell-Ash Company, Newton, Mass., a corporation of Massachusetts Filed Sept. 3, 1954, Ser. No. 454,182 4 Claims. (Cl. 235-92) This invention relates to the determination of data concerning discrete microscopic particles in a field of predetermined area.

While the invention is adapted for other purposes, it is herein discussed with particular reference to its use in hematology where the counting of red cells, for example, presents a series'of problems.

These problems may be understood with reference to standard visual techniques of blood counting. Such may be summarized as entailing the preparation of a specimen in which blood cells are suspended in a solution. With the specimen under suitable illumination, the technician counts the red cells within a preestablished rectangular field through a microscope. Apart from errors attributable to the usual causes of human failure, cells partly visible at the edges of the field present a problem of human judgment and represent a source of inaccuracies in the final count. The accepted practice is to count all the cells clearly within the rectangular field as well as those visible as overlapping two of its edges.

In the application for Letters Patent of Alan Richardson Jones entitled Scanning Instruments filed of even date herewith, now Patent No. 2,825,261, granted March 4, 1958, there is described and claimed apparatus well adapted for use in the fields with which this invention is concerned. In said application, the scanning instrument comprises a source of light, a precision aperture for beaming light emanating from that source on a photo-sensitive device, such as a photo-multiplier tube, and a stage for holding a specimen in the path of the light to provide a field in which particles are to be detected. Means are employed for moving the stage in a plane perpendicular to the path of the light with two dimensional components.

interposed between the field and the precision aperture.

is a microscope.

In the application of Frederick Brech entitled Method of and Apparatus for Use in Counting Pulses Stored in a..

Scale-r filed of even date herewith,,Serial No. 454,183,v filed September 3, 1954, there is described and claimed a method of and apparatus for use in visually presenting on a suitably calibrated dial the number of pulses of fixed amplitude stored in a scaler as a result of a test analysis. The invention of that application provides for the delivery to the scaler of a series of shaped electric pulses of fixed frequency after the test analysis until either limit of the capacity of the scaler, i.e., full or empty, is reached. Simultaneously, an electrically operatedtimer is driven synchronously with the fixed frequency pulses and, when the selected limit of the scaler is satisfied, its

discharge is used to terminate the timing. As the storage capacity of the scaling circuit and the number of pulses of. a fixed frequency delivered within the timed interval are both known, the diiference is the number of stored pulses and the timing device may be calibrated to enable such a result to be directly indicated.

.Such a direct reading or a determination by other suitable electronic means would, however, be in error due to the fact that edge cells along both edges of the field were indiscriminately counted. The principal objective of this invention is to eliminate material error from that source.

The present invention may be introduced by again reslot 28 is preferably of the order of one-half, more or' ferring to the application of Alan Richardson Jones wherein reference is made to a precision aperture through which the field is scanned and to the fact that such relative movement between the field and the aperture is provided as to establish a scanning pathway of predetermined length In fact, the pathway is reversible.

The present invention provides that in one scan an aperture is used that is in the form of a slot whose width is materially reater than that of the mean dimensions of the particle to be detected. In the second scanning along a pathway of the same length, the aperture is in the form of a slot whose width is less than that of said mean di- Scanning with the larger aperture in servicemensions. results in a reading including whole particles as well as edge particles on both edges. Scanning with the smaller aperture in service results in the counting only of edge particles and requires an approximate halving of the count which, when subtracted from the first total, ensures acceptable accuracy of the final reading in that,

in effect, whole particles plus those partly exposed on one edge only have been counted.

In the accompanying drawings, there is shown an illustrative embodiment of the invention from which this and other of its objectives, novel features and advantages will be readily apparent.

In the drawings:

FIG. 1 is a schematic view illustrating a part of an indicated pathway viewed through the aforementioned FIG. 4 is a fragmentary illustration of FIG. 3 with the smaller aperture operatively positioned.

FIGS. 5 and 6 illustrate, in association; with FIGS. 1',

and 2, the principles of edge cell correction, and

FIG. 7 is a schematic view of the counting circuits. In FIG. 3, there is indicated a scanning instrument for use in scanning the field containing the particles which hereinafter will be referred to as blood cells, these being suspended in a liquid to provide a speciment' 15 suitably,-

mounted on a carriage 16. The carriage 16 is movably mounted on a bed plate 17 with a dark field illuminator 18 being employed to illuminate the specimenlS. A

light tight box 19 has a microscope generally indicated at 20 and shown as equipped with a right-angle prism 21 thus to throw light on the photo-multiplier tube or-.-

other photo-sensitive surface 22 through the slot 23 in the element 24. The carriage 16 is recipr-ocated between two limits as by means of an eccentric 25 connected thereto by a rod 26 and rotated by the reversible motor 27. v

The scanning apparatus as above described illustrates general requirements and reference is made to said application of Alan Richardson Jones for a more detailed de scription of satisfactory scanning apparatus.

In accordance with the invention, and as shown in K FIGS. 3 and 4, the element 24 is also provided with a second slot 28 with the same length as but narrower than the slot 23. The slot 23 has a width preferably of the order of twice the projected diameter of cells en: countered in blood analysis, whereas the width of the less, the projected diameter of such cells. -In this connection, the mean diameter of blood cells of normal individuals may be'used as a standard.

It will be apparent that the cells detected during a g scan will fall into two categories when the slot dimension Patented Feb. 5, 1 963 is greater than one diameter. be:

(I) Cells lying wholly within the slot 23.

(II) Cells which are cut by or are touching either of the two edges thereof.

The proportion of the radius of cells of category II which may project within either slot 23 or 28 in order to be detected is not infinitely small since this quantity is dependent upon the level of sensitivity of the detecting device, which is, of course, finite.

Let it be assumed that the level of sensitivity is such that a finite area of the cells must project into the pathway in order for it to be detected. If a smaller area of the cell is presented, no detection will occur.

This means that the pathway limited by the slot 23 will include all the cells of category I and an unknown proportion of category II. For example, in FIG. 1 unshaded category II cells are detected, and the shaded ones are not. It will be apparent that the proportion of category II cells counted will determine the efiective width of the pathway as shown by the vertical lines in the diagram. Since it was impracticable to control precisely the sensitivity of the device and to influence the size of the cells, in most cases it is necessary to derive a correction for category II cells and subtract the corrected number of such cells present in the pathway from the total number previously detected (that is, category I plus II).

This is achieved by scanning the same field a second time over a pathway of the same length but with the slot 28 in position. Under these conditions, two new categories are derived as shown in FIG. 5:

(III) Cells cut by both edges of the slot.

(IV) Cells cut by one edge of the slot.

It is apparent that the sum of these categories must equal the number of cells in category II in the previous scan.

If now the number of cells detected with the use of slot 28 is divided by two and this figure is subtracted from the total count for the previous scan, the result will be equal to the number of cells effectively within the pathway whose width is equal to the difference between the width of the slot 23 and the width of the slot 28. The effect is to subtract from one edge of the slot 23 cells of category II and to retain as counted all cells of category I and the category II cells of the opposite edge, as shown in FIG. 6.

In order that such scaling procedures may be accomplished, it is preferable to utilize a circuit sequence illustrated in block form in FIG. 7.

Before discussing said circuit elements, reference is made to FIGS. 3 and 4 in which the element 24 is shown as a slide having a first position (see FIG. 3) in which the slot 23 is in beam receiving position, while slot 28 isnot. In FIG. 4, however, the position of these slots is reversed, shown as effected by the energizing of solenoid 29 in the circuit 30 provided with a switch 31 closed when the carriage 16 reaches the limit of travel of the first scan and remaining closed until the completion of the second scan.

During the course of the first scan, switch 31 remains Thus, the categories will open and therefore the binary circuit 32, for division bytwo, remains unenergized. Likewise, switch 33 at the opposing limit of travel of the carriage 16 to switch 31 remains open and therefore the fixed frequency pulse generator 34 remains unenergized. Accordingly, electrical signals generated in the photo-multiplier 22 by the image of cells viewed, in passing, through slot 23 are fed through an amplifier 35 into a shaping circuit 36 and from there pass onto a glow transfer decade sealer 37 in which the pulses are summed.

At the completion of the first scan, switch 31 is closed and remains so, thereby energizing the binary divider circuit 32. At the same time, the drive for the carriage is reversed and, as previously described, slot 28 replaces slot 23 in the beam receiving position.

At the same time, the direction of travel of the glow in the glow transfer tubes of the sealer 37 is reversed in a manner presently to be described thereby subtracting pulses from the previously summed total.

The binary divider circuit 32 is operatively connected to the amplifier 35. The circuit details of the binary divider 32 are well known and they are designed to provide a single out-put pulse for every two pulses provided as an input to it. Accordingly, the number of pulses summed in reverse scaling is half the number of pulses originating in the photo-multiplier 22.

In providing for a reversal of direction of the glow transfer tubes, an error is introduced in the subtraction phenomenon when cathode 10 is permitted to remain as the glow transfer position. This error is obviated when, on reversing the direction of the glow, cathode 9 is selected as the glow transfer position. Conveniently, such transfer may be achieved with the use of relays which will reverse the coupling to the succeeding tube from cathode 10 to cathode 9. This is illustrated by the chain dotted lines of FIG. 7 which have their counterpart in the corresponding full lines of the same FIGURE and which previous noted chain dotted lines come into play when switch 31 is opened with respect to the binary divider 32.

At the completion of the second scan, the carriage 16 closes the limit switch 33 and, at the same time, arrests the carriage drive thereby prohibiting further generation of signal pulses in the photo-multiplier 22. At the closure of switch 33, through a normally closed switch 38, a series of fixed frequency pulses are fed into the shaping circuit 36 which passes them into the scaling circuit 37. At the same time that the switch 33 is closed, the glow tube direction and the glow tube transfer position revert to their positions at the time of the first scan.

Simultaneously, with the commencement of fixed frequency pulse generation, an electrical timer 39 is started. The timer 39 is energized through a normally closed switch 40. Accordingly, the time taken for the fixed frequency pulses to fill the sealer 37 to its selected capacity from the previously summed value is an indication of that sum as described in said application of Frederick Brech in which the operation of the timing and fixed frequency pulse circuits is fully detailed.

What we therefore claim and desire to secure by Letters Patent is:

1. In a scanning instrument for detecting discrete microscopic particles such as blood cells, a support providing a field of predetermined area for the particles, means to illuminate said field, mechanism including microscope means having a first field viewing aperture in the form of a slot wider than the mean diameter of blood cells of normal individuals, and a second field viewing aperture in the form of a slot whose width is less than said diameter, means to effect relative movement between said support and said mechanism to effect the scanning of the field with first one aperture and then the other aperture in field-scanning position, each for approximately the same extent of such movement, means to generate electric Signals in response to the images of particles projected through said slots, and means to count and total such signals during the sequential scanning of said field through said slots, said last named means including a circuit, means connecting said circuit to said signal generatting means only during the scanning through the larger aperture, a second circuit including means converting a predetermined plurality of signals into a single signal, means connecting said second circuit to said signal generating means only during the scanning through the smaller aperture, a visual, reversible signal recorder, means connecting said recorder to whichever circuit is connected to said signal generating means, and means reversing the direction of said recorder when said second circuit is connected thereto.

2. In a scanning instrument for detecting discrete microscopic particles such as blood cells,a support providing a field of predetermined area for the particles, means to illuminate said field, mechanism including field viewing microscope means having a first field viewing aperture in the form of a slot wider than the mean diameter of blood cells of normal individuals and a second field viewing aperture in the form of a slot whose width is less than said diameter, means to efiect relative movement between said support and said mechanism to effect the scanning of the field with first one and then the other aperture in field scanning position, each for approximately the same extent of such movement, means to generate electric signals in response to the image of particles projected through said slots, means to count and total said electric signals during the sequential scanning of said field through said slots, said means including a circuit, means connecting said circuit to said signal generating means only during the scanning through the larger aperture, a binary circuit, means connecting said binary circuit to said signal generating means only during the scanning through the smaller aperture, a visual, reversible signal recorder, means connecting said recorder to whichever circuit is connected to said signal generating means, and means reversing the direction or" said recorder when said binary circuit is connected thereto.

3. In -a scanning instrument for detecting discrete microscopic particles such as blood cells, a support providing a field of predetermined area for the particles, means to illuminate said field, mechanism including microscope means having a first field viewing aperture in the form of a slot wider than the mean diameter of blood cells of normal individuals, and a second field viewing aperture in the form of a slot whose width is less than said diameter, said slots being of approximately the same length, reversible means to eflect relative movement between said support and said mechanism to eifect the scanning of the field along a predetermined pathway, means operable to substitute one aperture for the other in a field-scanning position at each end of said pathway, means to generate electric signals in response to the images of particles projected through said slots, and means to count and total such signals during the sequential scanning of said field through said slots, said last named means including a circuit, means connecting said circuit to said signal generating means only during the scanning through the larger aperture, is second circuit including means converting a predetermined plurality of signals into a single signal, means connecting said second circuit to said signal generating means only during the scanning through the smaller aperture, a visual, reversible signal recorder, means connecting said recorder to whichever circuit is connected to said signal generating means, and means reversing the direction of said recorder when said second circuit is connected thereto.

4. In a scanning instrument for detecting discrete microscopic particles such as blood cells, a support providing a field of predetermined area for the particles, means to illuminate said field, mechanism including microscope means having a first field viewing aperture in the form of a slot wider than the mean diameter of blood cells of normal individuals, and a second field viewing aperture in the form of a slot whose width is less than said diameter, said slots being of approximately the same length, reversible means to effect relative movement between said support and said mechanism to effect the scanning of the field along a predetermined pathway, means operable to substitute one aperture for the other in a field-scanning position at each end of said pathway, means to generate electric signals in response to the images of particles projected through said slots, means to count and total such signals during the sequential scanning of said field through said slots, said last named means including a circuit, means connecting said circuit to said signal generating means only during the scanning through the larger aperture, a second circuit including means converting a predetermined plurality of signals into a single signal, means connecting said second circuit to said signal generating means only during the scanning through the smaller aperture, a visual reversible signal recorder, means connecting said recorder to whichevercircuit is connected to said signal generating means, and means reversing the direction of said recorder when said second circuit is connected thereto.

References Cited in the file of this patent UNITED STATES PATENTS 2,661,902 Wolif Dec. 8, 1953 2,847,162 Meyer Aug. 12, 1958 2,850,239 Polanyi et a1. Sept. 2, 1958 7 OTHER REFERENCES Paper G. 3, The Automatic Counting of Red Blood Cells, by Cooke-Yarborough and Whyard, from The Physics of Particle Size Analysis, British Journal of Applied Sciences, Supplement No. 13, pages 5147-8156, published by The Institute of Physics, 47 Belgrave Square, London SW. 1, 

1. IN A SCANNING INSTRUMENT FOR DETECTING DISCRETE MICROSCOPIC PARTICLES SUCH AS BLOOD CELLS, A SUPPORT PROVIDING A FIELD OF PREDETERMINED AREA FOR THE PARTICLES, MEANS TO ILLUMINATE SAID FIELD, MECHANISM INCLUDING MICROSCOPE MEANS HAVING A FIRST FIELD VIEWING APERTURE IN THE FORM OF A SLOT WIDER THAN THE MEAN DIAMETER OF BLOOD CELLS OF NORMAL INDIVIDUALS, AND A SECOND FIELD VIEWING APERTURE IN THE FORM OF A SLOT WHOSE WIDTH IS LESS THAN SAID DIAMETER, MEANS TO EFFECT RELATIVE MOVEMENT BETWEEN SAID SUPPORT AND SAID MECHANISM TO EFFECT THE SCANNING OF THE FIELD WITH FIRST ONE APERTURE AND THEN THE OTHER APERTURE IN FIELD-SCANNING POSITION, EACH FOR APPROXIMATELY THE SAME EXTENT OF SUCH MOVEMENT, MEANS TO GENERATE ELECTRIC SIGNALS IN RESPONSE TO THE IMAGES OF PARTICLES PROJECTED THROUGH SAID SLOTS, AND MEANS TO COUNT AND TOTAL SUCH SIGNALS DURING THE SEQUENTIAL SCANNING OF SAID FIELD THROUGH SAID SLOTS, SAID LAST NAMED MEANS INCLUDING A CIRCUIT, MEANS CONNECTING SAID CIRCUIT TO SAID SIGNAL GENERATING MEANS ONLY DURING THE SCANNING THROUGH THE LARGER APERTURE, A SECOND CIRCUIT INCLUDING MEANS CONVERTING A PREDETERMINED PLURALITY OF SIGNALS INTO A SINGLE SIGNAL, MEANS CONNECTING SAID SECOND CIRCUIT TO SAID SIGNAL GENERATING MEANS ONLY DURING THE SCANNING THROUGH THE SMALLER APERTURE, A VISUAL, REVERSIBLE SIGNAL RECORDER, MEANS CONNECTING SAID RECORDER TO WHICHEVER CIRCUIT IS CONNECTED TO SAID SIGNAL GENERATING MEANS, AND MEANS REVERSING THE DIRECTION OF SAID RECORDER WHEN SAID SECOND CIRCUIT IS CONNECTED THERETO. 